This invention relates to plant tissue culture media designed to more efficiently obtain transgenic plant cells from Agrobacterium-mediated transformation, and more particularly to plant transformation media containing an effective amount of a nitric oxide modulator.
The ability to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology has become widespread in recent years. This advance has provided enormous opportunities to improve plant resistance to pests, disease and herbicides, and to modify biosynthetic processes to change the quality of plant products. A highly efficient method for transformation of these crop plants continues to be a goal as there is a need for high capacity production of economically important plants.
Agrobacterium-mediated transformation is one method for transforming such crop plants and has more recently become more adaptable for use in monocotyledonous plants. Several Agrobacterium species mediate the transfer of a specific DNA known as “T-DNA” that can be genetically engineered to carry a desired piece of DNA into the selected plant species. The major events marking the process of T-DNA mediated pathogenesis and ultimately transformation are induction of virulence genes, processing and transfer of the T-DNA to the plant's genome.
Typically, Agrobacterium-mediated genetic transformation of plants involves several steps. The first step, in which the Agrobacterium and plant cells are brought into contact with each other, is generally called “inoculation.” Following the inoculation step, the Agrobacterium and plant cells/tissues are usually grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer. This step is termed “co-culture”. Following co-culture and T-DNA delivery, the plant cells are often treated with bactericidal or bacteriostatic agents to prevent further growth of the Agrobacterium. If this is done in the absence of any selective agents to promote preferential growth of transgenic versus non-transgenic plant cells, then this is typically referred to as the “delay” step. If done in the presence of selective pressure favoring transgenic plant cells, then it is referred to as a “selection” step. When a “delay” is used, one or more “selection” steps usually follow it. Both the “delay” and “selection” steps typically include bactericidal or bacteriostatic agents to prevent further growth of any remaining Agrobacterium cells because the growth of Agrobacterium cells is undesirable after the infection (inoculation and co-culture) process.
Modulation of the plant response to Agrobacterium is an important part of Agrobacterium transformation, because Agrobacterium transformation is a variation of the disease process caused by Agrobacterium. One of the variables is getting the right amount of infection to cause efficient transformation without too much infection overwhelming the plant and doing damage.
In plants, nitric oxide (NO) has been implicated in disease resistance and various types of defense responses to environmental stresses (Leshem et al., Plant Physiology Biochemistry 35: 573-579, 1997; Plant Physiology and Biochemistry 36: 825-833, 1998; Millar and Day, Trends in Plant Science 2: 289-290, 1997; Klessig, Proceedings National Academy of Sciences USA 97: 8849-8855, 1999; Magalhaes et al., Physiology Molecular Biology Plants 5: 115-125, 1999; Pedroso et al., Plant Science 157: 173-180, 2000; Pedroso et al., Journal of Experimental Botany 51: 1027-1036, 2000; Garces et al., Annals of Botany 87: 567-574, 2001). In animals, NO is produced from L-arginine by both constitutive and inducible NO-synthases (NOS) (Garvin, In: Sitaramayya A. ed., Introduction to Cellular Signal Transduction. Boston. pp 177-212, 1999). A similar pathway may exist in plants because NO production inhibitors used with animal cells also inhibit NO production in plants (Guo et al., Science 302: 100-103, 2003). NO can also be generated in plants as a by-product of nitrate reductase, nitrogen fixation, or respiration (Klepper, Plant Physiology 93: 26-32, 1990; Norditake et al., Plant Cell and Physiology).
Because NO has been implicated in defense responses, its control or elimination may increase pathogen infection such as that of Agrobacterium during plant transformation. The present invention provides a novel method of increasing Agrobacterium-mediated transformation through the use of compounds and processes that modulate endogenous NO levels.